Summary of the Invention
This invention relates to a novel use for a bacteriocin and/or the bacteria that produces this bacteriocin. The novel use involving killing Streptococcus pyogenes, Streptococcus mutans, and/or Propionibacterium acnes, and preventing or treating diseases caused by these bacteria. This invention also relates to compositions containing this bacteriocin and/or the bacteria that produce this bacteriocin useful in preventing or treating diseases caused by S. pyogenes, S. mutans, and/or P. acnes and killing S. pyogenes, S. mutans, and/or P. acnes. 
Summary of the Prior Art
Streptococcus thermophilus is a thermophilic lactic acid bacteria commonly used as a starter culture in the production of yogurt and cheeses. Several strains of S. thermophilus have been reported to naturally produce antimicrobial peptides called bacteriocins. These ribosomally encoded peptides have been shown to display a narrow and/or broad spectrum of activity with the potential to inhibit the growth of spoilage and food-borne pathogenic bacteria (Villani, et al., Int. J. Food Microbiol. 25, 179-90 (1995); Ward & Somkuti, Appl. Microbiol. Biotechnol. 43, 330-5 (1995); Marciset, et al., J. Biol. Chem. 272, 14277-84 (1997); Ivanova, et al., Int. J. Food Microbiol. 42, 147-58 (1998); Mathot, et al., J. Dairy Sci. 86, 3068-74 (2003); Gilbreth & Somkuti, Curr. Microbiol. 51, 175-82 (2005); Kabuki, et al., J. Appl. Microbiol. 102, 971-80 (2007); Khalil, Pol. J. Microbiol. 58, 49-55 (2009); Rossi, et al., Food Microbiol. 35, 27-33 (2013)). The genes encoding these bacteriocins have been identified for thermophilin 13, a two component bacteriocin (Marciset, et al. (1997)), and the lantibiotic thermophilin 1277 (Kabuki, et al. (2007)); but remain uncharacterized in other S. thermophilus strains.
Comparative genomic studies on S. thermophilus strains LMD-9, CNRZ1066 and LMG18311 (Hols, et al., FEMS Microbiol. Rev. 29, 435-63 (2005)), which were initially thought to not produce a bacteriocin, revealed the presence of a gene cluster resembling the class II bacteriocin-like peptide locus in Streptococcus pneumoniae (de Saizieu, et al., J. Bacteriol. 182, 4696-703 (2000)). Each S. thermophilus strain was shown to contain a three gene operon (blpABC) that encoded the components of an ABC-transporter (blpAB) required for processing and secretion of a quorum-sensing induction peptide (QSIP), encoded by blpC. The QSIP was initially expressed as a 53 residue prepeptide and further processed to the mature 30-mer QSIP by removal of a 23 amino acid leader peptide (Fontaine, et al., J. Bacteriol. 189, 7195-205 (2007)). Immediately downstream of blpABC, was a two gene operon (blpRH) that encoded a histidine kinase (BlpH) and response regulator (BlpR) required for sensing the QSIP and inducing the expression of other genes within the cluster (Blomqvist, et al., Arch. Microbiol. 186, 465-73 (2006)). The remaining components of each gene cluster differed significantly with S. thermophilus strains LMD-9, LMG18311 and CNRZ1066 possessing 15, 12 and 4 genes respectively. Within this region were the genes believed to encode the actual bacteriocins based on the presence of a double glycine leader sequence and included blpD, blpU, blpE and blpF in S. thermophilus strain LMD-9; blpU and blpK in S. thermophilus strain LMG18311 and blpK in S. thermophilus strain CNRZ1066 (Hols, et al. (2005)). In S. thermophilus strain LMD-9, the addition of synthetic QSIP to the growth medium resulted in the expression of a broad spectrum bacteriocin (Fontaine, et al. (2007)), but it failed to induce bacteriocin production in S. thermophilus strains LMG81311 and CNRZ1066. The inability of these two strains to produce an active bacteriocin was thought to result from a truncation in BlpB which is required for the secretion of the antimicrobial peptides (Hols, et al. (2005)). Further analysis of S. thermophilus strain LMD-9 revealed that BlpD alone was sufficient to inhibit the growth of most target bacteria, thus BlpU, BlpE, BlpF were classified as accessory peptides (Fontaine & Hols, Appl. Environ. Microbiol. 74, 1102-10 (2008)). In S. thermophilus strain LMD-9, a thiol-disulfide oxidase encoded by blpG was identified, and shown to be essential for the strain's novel anti-listerial activity (Fontaine & Hols (2008)). Additional variations within this gene cluster have been reported in other S. thermophilus strains (Rossi, et al. (2013)), and the potential for identifying new variants increases as more S. thermophilus genomes are fully sequenced.
Previously, the natural production of an anti-pediococcal bacteriocin, thermophilin 110, by S. thermophilus strain 110 (USDA, ARS NRRL patent depository accession number B59671) (hereinafter referred to as “S. thermophilus strain 110 (NRRL B59671)”) was described (Gilbreth & Somkuti (2005)). Another study reported that thermophilin 110 production was regulated by BlpC (Renye & Somkuti, Biotechnol. Lett. 35, 407-12 (2013)).
Herein, it is demonstrated that pro-thermophilin 110 produced by S. thermophilus strain 110 (NRRL B59671) is encoded by blpU. The pro-thermophilin 110, also referred to as BlpU, has the amino acid sequence of SEQ ID NO: 1. This sequence includes a 23 amino acid leader sequence (SEQ ID NO: 38; amino acids 1-23 of SEQ ID NO: 1) that is cleaved off during translation or post-translational processing. The amino acid sequence of mature thermophilin 110 protein is in SEQ ID NO: 36 and is also amino acids 24-76 of SEQ ID NO: 1. The DNA sequence for blpU is in SEQ ID NO: 2 which includes nucleotides 1-69 that encode for the 23 amino acid leader sequence peptide. The DNA sequence of the mature thermophilin 110 protein is in SEQ ID NO: 37 and is also nucleotides 70-228 of SEQ ID NO: 2. As demonstrated infra, mature thermophilin 110 has anti-bacterial activity. Pro-thermophilin 110 lacks anti-bacterial activity. As such, when used herein, “thermophilin 110” refers to mature thermophilin 110 (amino acids 24-76 of SEQ ID NO: 1 which is the same as all of SEQ ID NO: 36), unless the context clearly indicates the pro-thermophilin 110 form.
Also demonstrated herein, thermophilin 110 has anti-bacterial activity against S. pyogenes, S. mutans, and P. acnes; this anti-bacterial activity being novel and unknown prior to the studies reported herein. Furthermore, no other bacteriocin made by S. thermophilus has exhibited bactericidal activity against S. pyogenes, S. mutans, and P. acnes. S. thermophilus strain 110 (NRRL B59671) can be used a probiotic composition to prevent the growth of and/or kill S. pyogenes, S. mutans, and P. acnes, and thus prevent, reduce the symptoms of, and/or treat the diseases caused by these bacteria, namely strep throat, dental caries, and acne, respectively. P. acnes is also suspected to be involved in post-operative infections, prostheses failure, and in inflammation of lumbar nerve roots leading to sciatica.